Ordering Information

pCLuc Mini-TK 2 is a cloning vector for
mammalian cells, containing a minimal promoter
fragment from the HSV thymidine kinase (TK)
promoter adjacent to a reporter gene, the secreted
luciferase from the Ostracod Cypridina noctiluca.
Cypridina luciferase (CLuc) is a 62 kDa protein
with a native signal peptide at the N-terminus that
allows it to be secreted from mammalian cells (1)
so that CLuc activity can be detected in the culture
medium of mammalian cells expressing the reporter
gene. The pCLuc Mini-TK 2 Vector contains a MCS
upstream of the minimal TK promoter for cloning
promoter or enhancer elements. A neomycin resistance
gene under the control of an SV40 promoter
allows selection for stable integration of the plasmid
into the mammalian cell genome using G418.

DNASU and Addgene are central repositories for plasmid clones and collections that may also be helpful.

Highlights

Multiple samples can be obtained from the same
transfected cells (i.e., before and after experimental
treatments or at multiple time points).

90–95% of CLuc activity is found in the cell
culture medium, with the remaining 5-10%
detectable in cell lysates (Figure 1). This allows
flexibility when assaying CLuc along with other
co-transfected reporters.

The activity of CLuc is high and the CLuc assay is
sensitive enough to detect very small amounts of
CLuc enzyme activity (Figure 2).

CLuc does not use the same substrate as other
marine luciferases (e.g. Renilla, Gaussia). Therefore,
it is possible to assay both CLuc and GLuc
independently in cell culture medium from cells
expressing both reporters.

The pCLuc Mini-TK 2 Vector can be transfected into cells using any standard transfection protocol.

Two restriction sites are available for cloning elements downstream of the CLuc coding region. The NotI site is upstream of the polyA site and allows cloning of sequences which will become part of the CLuc mRNA. The XbaI site is downstream of the polyA site, sequences cloned into the XbaI site will not be incorporated into CLuc mRNA.

All pLuc-2 vectors have improved poly-adenylationtranscription termination of the luciferase transcript. The polyadenylation signal is a synthetic polyadenylation sequence based on the b-globin gene (5).

Application Features

The pCLuc Mini-TK 2 Vector can be used to test promoter or enhancer elements by cloning into the MCS upstream of the minimal TK promoter. For constitutive expression of CLuc, vectors containing constitutive promoter elements are available (see Related Products).

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Licenses

Licensed under certain patents and patent applications from the National Institute of Advanced Industrial Science and Technology ("AIST") for Research and Development Purposes.

For use of the Biolux Cypridina Luciferase Assay Kit, or associated assay reagents, in human diagnosis and measurement in relation to human health, contact busdev@neb.com.

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